When electric field is supplied to a charged substance in an electrolyte solution, the substance migrates toward the electrode having an opposite charge of the substance. This phenomenon, electrophoresis, is widely used as a means for separating various substances. Generally, electrophoresis of an analytical sample is performed in a carrier having a constant pH. On the contrary, a carrier having a gradient pH is used when electrophoresis is performed based on the isoelectric focusing method. Since the isoelectric focusing method was developed, it has been acquiring popularity as a means for separating amphoteric electrolytes, such as amino acids and proteins.
An amphoteric electrolyte has a pH value where its effective charge becomes zero, and the pH value is called an isoelectric point. When electrophoresis of the analytical sample consisting of an amphoteric electrolyte is performed based on the isoelectric focusing method, the sample stops at a certain position in an electrophoresis carrier and is concentrated there. This position is where a pH value of the electrophoresis carrier is equal to the isoelectric point of the sample. In this case, the separated sample is concentrated in a focusing manner, therefore, the isoelectric focusing method has very high separability.
Recently, the capillary electrophoresis, where electrophoresis is performed in a capillary having an inside diameter of several 10 micrometers and a length of several 100 millimeters, was established. Using this method, very high separability is obtained with a very small amount of analytical sample. Therefore, the method is applied to separation and analysis of various samples including proteins, inorganic ions, low molecular compounds, nucleic acids, and the like. When the capillary electrophoresis is performed, concentration of the separated analytical sample can be quantified using a detector equipped at one end of the capillary.
Detection of the sample separated by the electrophoresis is generally done by the ultraviolet/visible detecting method that includes irradiation of an ultraviolet light or a visible light to the sample and measurement of the changes of the amount of light absorbed. Detection can be done with higher sensitivity when a fluorescence detection method is applied. In the fluorescence detection method, an analytical sample is labeled with a fluorescent dye (fluorescently labeling). In this method, an excitation light is focused on the separated sample, and fluorescence generated is detected to quantify the concentration of the sample.
It is possible to combine above-mentioned isoelectric focusing method, capillary electrophoresis, and fluorescence detection method. The combined method is called fluorescently detecting capillary isoelectric focusing. In the case of fluorescently detecting capillary isoelectric focusing, electrophoresis is performed to the fluorescently labeled sample in a carrier having a gradient pH, which is held in the capillary, and the fluorescence caused by irradiation of the excitation light is detected with an optical detector or the like. According to this method, even with the sample of a very small amount, a highly precise quantitative detection is possible. Therefore, this method attracts attention as a super-high-sensitive analytical method for proteins or the like.
In recent years, a minor constituent in a living body is analyzed by the electrophoresis mentioned above in many cases. When performing such analysis, an immune complex formed by reacting a minor constituent in a living body with the antibody that recognizes the minor constituent as an antigen is detected. The immune complex is preferrably labeled with a fluorescent dye for the purpose of accurate detection. In this case, either the antigen or the antibody needs to be fluorescently labeled. Upon labeling the antigen or the antibody using the fluorescent dye, conventional labeling method can not be applied due to the following reasons.
Antibodies and many of antigens are composed of proteins. The number of amino groups at an N-terminal of a protein and at a lysine side chain, and the dissociated state thereof are a great factor for determining an isoelectric point of a protein (Zokuseikagaku Jikkenkoza 2, Chemistry of proteins, the volume 1, Society of Japan Biochemistry, 1987). Therefore, the conventional labeling method utilizing a reaction between a fluorescent dye and an amino group of a protein greatly changes an isoelectric point of a protein.
In addition, the number and a position of a fluorescent dye bound to a protein become indefinite because there are a large number of amino acids that are reactive with a fluorescent dye in a protein. Accordingly, the resultant protein becomes a mixture of proteins showing different isoelectric point, which makes it difficult to conduct a precise analysis using the isoelectric focusing. Further, since the three-dimensional structure of a protein is changed by a fluorescent dye, there is also a problem that the chemical stability of a protein itself is deteriorated.
In addition, there is a case that an analysis by the isoelectric focusing can not be done with high accuracy even when a monoclonal antibody having a uniform molecular weight obtained by a hybridoma is used as an antibody for detection. This is because the monoclonal antibody having a uniform molecular weight produced by hybridoma does not necessarily have a uniform isoelectric point, and this phenomenon is called microheterogeneity (Bouman H et al. Z Immunitatsforsch Exp Klin Immunol. 1975 October; 150(4): 370-7).
As a reason for this ununiformity of an isoelectric point, there have been proposed deamidation of a protein (Robinson A. B. et al., Proc. Natl. Acad. Sci. U.S.A. 1970 July; 66(3): 753-7), pyroglutamylation of an N-terminal (Scott D. I. et al., Biochem. J. 1972 August; 128(5): 1221-7), addition of a sugarchain (Cohenford M. A. et al. Immunol. Commun. 1983; 12(2): 189-200), myristoylation (Pillai S. et al., Proc. Natl. Acad. Sci. U.S.A. 1987 November; 84(21): 7654-8) and the like, but the mechanism for the ununiformity of an isoelectric point of a protein has not been specified yet.
Therefore, when an analytical sample shows plural isoelectric points by isoelectric focusing, it is difficult to determine where the plurality is originated from because the plurality is ascribable either to an antigen or to an antibody. This is because both antigen and antibody may have ununiformity of an isoelectric point as mentioned above.
Therefore, when analysis is performed by electrophoresis utilizing an antigen-antibody reaction, an antibody is necessary to be uniform in terms of isoelectric point, and when an antibody having a uniform isoelectric point is fluorescently labeled with a fluorescent dye, the fluorescent dye should not be reacted with an amino group of the antibody as described above.
As a method for quantitatively detecting an antigen using an antibody having a uniform isoelectric point, a method of Shimura K. and Karger B. L. is known (see Anal. Chem. 1994 Jan. 1; 66(1):9-15, or JP-A 8-506182). The method disclosed in these references is schematically shown in FIGS. 8A to G. That is, IgG antibody produced by a hybridoma (FIG. 8A) is cut with a protease (pepsin) and the resulting F(ab′)2 antibody (FIG. 8B) is separated. This is treated with a reducing agent of mercaptoethylamine to reduce three disulfide bonds (S—S bond) to obtain Fab′ antibody (FIG. 8C). This Fab′ antibody is oxidized to make only one reactive thiol group (SH group) left (FIG. 8D) and a fluorescent dye is bound to this thiol group (FIG. 8E). The resulting fluorescently labeled Fab′ antibody is separated by the isoelectric focusing and a fluorescently labeled Fab′ antibody having a uniform isoelectric point is taken out from an electrophresis carrier (FIG. 8F). The obtained fluorescently labeled Fab′ antibody having a uniform isoelectric point is combined with an antigen. Then, electrophoresis is performed, and fluorescence caused by excitation light is measured (FIG. 8G).